Virus-host interactions of tomato yellow ring virus, a new tospovirus from Iran

During the past decades, an increasing number of new tospovirus species occurring in various agricultural and horticultural crops have been reported. The emergence of new tospoviruses may be attributed to intensified international trading, to increasing problems to control their thrips vectors, but certainly also by better recognition based on new diagnostic tools.
The works presented in the thesis first focused on comprehensive characterisation and identification of a tospovirus species occurring in different crops in Iran, and next on transgenic approaches to control this virus.
In Chapter 2, five presumed Iranian tospovirus isolates from tomato, chrysanthemum, gazania, soybean and potato, collected in 2002, were analysed. All isolates induced necrotic local lesions on Petunia hybrida, indicative for tospoviruses. None of the available antisera against known tospoviruses reacted with the isolates, suggesting that if these were tospoviruses, they should belong to a novel species. As a next step the viral nucleoprotein (N) gene of the tomato isolate was cloned and sequenced and this information demonstrated that it represented a new tospovirus species for which the name Tomato yellow ring virus (TYRV) was proposed. Subsequently the N gene sequences of the chrysanthemum and gazania isolates were also obtained and showed these isolates to represent TYRV as well. Back-inoculation of the tomato isolate induced resembling chlorotic and necrotic spots on leaves and yellow rings on the tomato fruits. The complete S RNA sequence of this isolate revealed the generic topology of a tospoviral S RNA, containing both NSS (suppressor of silencing) and N (nucleocapsid) genes separated by a long non-coding intergenic region including a predicted hairpin structure. Multiple sequence alignment of the N protein of TYRV with those of established tospovirus species revealed the closest relationship (74% identity) to Iris yellow spot virus (IYSV).
In chapter 3, the isolates from soybean and potato were analysed. Although in ELISA assays these isolates scored positive with antiserum raised against the TYRV-tomato isolate, they failed to amplify in RT-PCR when using primers derived from the latter. The N gene sequences of these isolates indeed revealed a sequence divergence of 8% compared to that of TYRV-tomato, indicating these two belonged to different strains of TYRV (denoted TYRV-s, while the strain occurring in tomato was named TYRV-t). Additional differences between the two strains were found in their respective S RNA non-coding intergenic regions. Differences in their host range and symptom expression underscored the decision to treat them as separate strains. A preliminary cross-protection study indicated that TYRV-t and TYRV-s mutually exclude each other, indicating that the strains represent stable, isolated lineages which do not easily converge despite their geographical overlap.
In chapter 4, extended inverted repeat transgenic cassettes for broad tospoviral resistance were constructed and tested. These transgene cassettes contained partial N gene sequences from 5 different tomato-infecting tospoviruses, including TYRV-t from Iran, in such arrangement that transgenic expression would deliver a ds hairpin RNA. Using Nicotiana benthamiana as a model plant, transgenic lines harboring an inverted repeat construct interspaced with a sense-oriented intron were obtained with high frequencies of resistance up to 100% for all 5 tospoviruses in F2. By analysing the siRNA content of the transgenic plants it could be verified that the transgenic resistance was based on RNA silencing (or shortly RNAi). Whilst these transgenics were fully resistant to TYRV-t (whose N gene sequence was used in the transgene cassette), they were fully susceptible to TYRV-s, demonstrating again how narrow transgenic resistance based on RNAi is. Surprisingly upon co-inoculation with TYRV-s, TYRV-t also could overcome the transgenic resistance. Mass spectrometric analysis of viral ribonucleocapsid protein (RNP) purified from a mixed-infected transgenic line revealed that the N protein of both strains were present and hence hetero-encapsidation as possible mechanism to rescue TYRV-t from these plants could be excluded. Experiments involving the expression of the viral suppressor protein (NSs) from TYRV-s using a PVX vector, indicated that rescue of TYRV-t by TYRV-s was based on the expression of the TYRV-s RNAi suppressor.
Since most of the operational transgenic tospovirus resistance approaches are based on RNAi, involving the production of transgenic viral siRNAs, in chapter 5 the production and involvement of viral siRNA molecules during a natural tospovirus infection process was investigated. Special attention was given to the intergenic hairpin region of the S RNA segment. Total small RNA was isolated from TYRV-t infected N. benthamiana and mapped to the S RNA segment. These studies demonstrated the occurrence of a hot spot for siRNA induction within the S RNA, but surprisingly this hotspot was not mapped in the IGR hairpin but at the start of NSS ORF where a much shorter hairpin structure was predicted. Surprisingly, fewest siRNAs mapped to the intergenic region which is predicted to fold into a long hairpin, despite additional experiments involving DICER cleavage by Drosophila melanogaster embryo extract and YFP (yellow fluorescent protein)-hairpin constructs indicating this region to be a potential inducer and target for RNAi.
In chapter 6, the observations done in the experimental chapters are discussed in a broader context and a model for tospovirus-induced RNA silencing presented.